Lectin‐binding patterns of small lymphocytes in bone marrow, thymus and spleen: demonstration of lymphocyte subsets by quantitative radioautography

Saveriano, N; Drinnan, Michael; Santer, Vivien; Osmond, Dennis G.

doi:10.1002/eji.1830111105

Cited by 13 publications

(5 citation statements)

References 27 publications

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“…Axelsson et al [7] and Haller et al [8] found it necessary to remove sialic acid to bind HPA to spleen cells. On the other hand, other authors reported the presence of 1 5 4 0 % of HPA-positive cells in untreated mouse spleen cell samples, as detected by autoradiography [34] or rosetting technique [35]. The present observations are more consistent with the first group of authors when strict criteria for membrane staining are applied.…”

Section: Discussionsupporting

confidence: 93%

Relationship between mouse lymphocyte receptors for peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA)

Petris

Takács²

1983

Eur J Immunol

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The relationship between mouse lymphocyte receptors for peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA) has been investigated by immunofluorescence (cocapping) and radiolabeling. In neuraminidase-treated and untreated thymocytes there are two groups of glycoproteins which bind roughly equivalent amounts of PNA. One group also carries all the detectable receptors for HPA, the other binds only PNA. Binding inhibition experiments suggest that PNA and HPA receptors are in close proximity on the shared glycoproteins. The same two groups of receptors are present on 35-40% of neuraminidase-treated spleen lymphoid cells, mainly immunoglobulin (Ig)-negative lymphocytes. Almost all B cells have only PNA-specific receptors. Five-12% of the untreated spleen cells appreciably bind PNA and only a few bind HPA. Solubilized glycoproteins specific for PNA or HPA were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The major PNA-specific radioiodinated glycoproteins of neuraminidase-treated thymocytes, as isolated by affinity chromatography, consist of the 185-kDa and 195-kDa components of the T200 antigen and of two (diffuse) components of about 140 and 120-125 kDa. All these molecules also bind to HPA-Sepharose, with the exception of the 185 kDa component, which is probably the main constituent of the "pure" PNA receptors on the intact thymocytes. In gels directly labeled with radioactive lectins, the only band strongly labeled by PNA and HPA is the diffuse 140-kDa band. The band at 120 kDa is well labeled by PNA, but all the other components are weakly labeled. The mobility of the 140- and 120-kDa bands depends strongly on neuraminidase-treatment. These bands cannot be detected in gels of untreated thymocytes, but a major HPA-and PNA-specific band of lower molecular weight can be labeled after treating the gels with neuraminidase. The factors determining the differences in labeling pattern obtained by different methods as well as the nature of PNA and HPA binding sites are discussed. The same major PNA- and HPA-binding glycoproteins (apart from minor differences) are present on neuraminidase-treated Ig-negative spleen lymphocytes. The major PNA-binding protein of B lymphocytes appears to correspond to the 225-kDa ("B220") antigen specific for these cells.

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Section: Discussionsupporting

confidence: 93%

Relationship between mouse lymphocyte receptors for peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA)

Petris

Takács²

1983

Eur J Immunol

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“…Of the sy' small lymphocytes in the BM only 1/10 bound readily detectable amounts of PNA, in agreement with doubleimmunofluorescence labeling [9], these cells having the lowest density of surface IgM and thus being the earliest of the sp' cells in the maturation pathway (Scheme 1). In general, sp' small lymphocytes in the spleen do not bind PNA detectably [14, 221 even when exposed to high concentrations of the radiolabeled lectin and analyzed by sensitive radioautographic techniques [16]. The few Ig-bearing cells in the spleen which do bind some PNA also show a negative correlation between PNA binding and sp density [14], and may thus be recent immigrants from the BM.…”

Section: Discussionmentioning

confidence: 99%

“…Combinations of lectins, however, representing several saccharide specificities, may define finer subsets of cells by their labeling pattern [15,161. Among a panel of seven lectins tested, wheat germ agglutinin (WGA) was distinctive in binding preferentially to BM lymphocytes [16] including cp'sp-cells [25]. Unlike PNA, on the other hand, WGA also binds strongly to sp' B lymphocytes [15, 251 but shows no preferential binding to colony-forming cells [15].…”

Section: Discussionmentioning

confidence: 99%

“…In the BM, immunofluorescence labeling has revealed the binding of PNA to cp'sp-pre-B cells [9, 141 but to only a minority of the nonlymphoid hemopoietic cells [15]. In a radioautographic study of the binding of a panel of lectins of various specificities PNA was unique in binding to a restricted subset of BM lymphocytes [16], while with few exceptions it failed to bind to mature small lymphocytes in the spleen [14, 161. Of the lectins examined, PNA thus promises to provide a useful means of further characterizing the BM B cell precursors.…”

Section: Pre-b Cells In Bone Marrow: Peanut Agglutinin Binding and Sementioning

confidence: 99%

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Pre‐B cells in bone marrow: peanut agglutinin binding and separation of cytoplasmic μ chain‐bearing cell populations in normal, post‐irradiation and polycythemic mice using fluorescence‐activated cell sorting

Osmond

1984

Eur J Immunol

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Mouse bone marrow cells exposed to fluorescein-conjugated peanut agglutinin (PNA) showed subsets of highly labeled cells when analyzed in a fluorescence-activated cell sorter. After separating three cell fractions of large and small PNA-binding cells and PNA-nonbinding cells, respectively, the B lymphocyte precursor (pre-B) cells, having cytoplasmic mu chains (c mu) without surface mu chains (s mu), were recovered solely in the PNA-binding fractions. Only a minority of s mu+ small lymphocytes having the lowest densities of s mu bound PNA. Small and large c mu+ s mu- pre-B cell populations were separated in high degrees of purity in the PNA-binding fractions, especially when obtained from bone marrow undergoing lymphoid regeneration after sublethal X-irradiation and during stimulation of lymphocyte production in post-polycythemic erythroid suppression. Characteristic shifts in the size distribution profile of PNA-binding cells reflected changes in the maturation stage of the pre-B cells. The results demonstrate that surface membrane components with strong PNA-binding capacities characterize c mu+ s mu- pre-B cells in the bone marrow during both normal and perturbed primary B lymphocyte genesis. The PNA-binding sites become undetectable soon after the first expression of s mu. This property permits the isolation from the bone marrow of high concentrations of subsets of large and small c mu+ s mu- cells in a viable state suitable for use in further functional studies.

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“…The cells were then fixed in 1.5% glutaraldehyde in PBS, pH 7.4, for 1 h at 4"C and washed twice in PBS before labeling. A suspension ofthymic cells from the pooled thymi of six mice (C3H, 10-wk old) was prepared (25) and the cells were incubated at 37"C for 30 min and fixed as above. Samples of sperm from mature boars, Sus scrofa (a gift of Dr. Larry Johnson of the U. S. Department of Agriculture, Beltsville, MD), were washed three times in PBS or in Hank's balanced salt solution (19), fixed, and washed as above.…”

Section: Materials a N D M E T H O D Smentioning

confidence: 99%

Label-fracture: a method for high resolution labeling of cell surfaces.

Silva¹,

Kan²

1984

The Journal of Cell Biology

124

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We introduce here a technique, "label-fracture," that allows the observation of the distribution of a cytochemical label on a cell surface. Cell surfaces labeled with an electron-dense marker (celloidal gold) are freeze-fractured and the fracture faces are replicated by plantinum/carbon evaporation. The exoplasmic halves of the membrane, apparently stabilized by the deposition of the Pt/C replica, are washed in distilled water. The new method reveals the surface distribution of the label coincident with the PtIC replica of the exoplasmic fracture face.Initial applications indicate high resolution (~<15 nm) and exceedingly low background. "Labelfracture" provides extensive views of the distribution of the label on membrane surfaces while preserving cell shape and relating to the freeze-fracture morphology of exoplasmic fracture faces. The regionalization of wheat germ agglutinin receptors on the plasma membranes of boar sperm cells is illustrated. The method and the interpretation of its results are straightforward. Label-fracture is appropriate for routine use as a surface labeling technique.The localization of cell-surface receptors, antigens, and other chemical groups associated with lipid or protein molecules of the plasma membrane is a recognized objective of the cell sciences. Ultrastructural cytochemistry of cell surfaces was first observed in thin sections of cells and tissues, a process that required the assemblage of composites obtained from serial-sectioned cells (1). To overcome this unusually difficult procedure, other techniques were developed that allowed direct views of the surface distribution of the label: (a) plasma membranes collapsed at an air/water interface (2, 3); (b) platinum/carbon replication of cells (air-dried, freeze-dried, or critical-point dried) (4); and (c) platinum/carbon replication of frozen cells after freeze-fracture and sublimation (freeze-etching) (5-9). All these approaches suffered from severe limitations. Collection of unfixed (or lightly fixed) membranes at the air/water interface could not prevent reorganization of receptors (10). Conventional Pt/C replication of dried specimens could only be applied to cell monolayers (or to cells previously attached to a substrate), and drying procedures could cause deformation and collapse of structures (11, 12). Freeze-etching related the surface distribution of receptors and antigens to that of the intramembrane particles revealed by freeze-fracture but could only be performed in systems (generally isolated membranes) capable of withstanding freezing in the absence of cryoprotectants.Over the past few years, we have developed new labeling techniques--fracture-label (13-16)--that allow direct, in situ labeling of freeze-fractured plasma and intracellular membranes. As freeze-fracture splits biomembranes along their bilayered continuum (17, 18), fracture-label techniques can be used to determine the sidedness of membrane components and, in some instances, to identify transmembrane proteins as well as their regionalization ...

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Lectin‐binding patterns of small lymphocytes in bone marrow, thymus and spleen: demonstration of lymphocyte subsets by quantitative radioautography

Cited by 13 publications

References 27 publications

Relationship between mouse lymphocyte receptors for peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA)

Relationship between mouse lymphocyte receptors for peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA)

Pre‐B cells in bone marrow: peanut agglutinin binding and separation of cytoplasmic μ chain‐bearing cell populations in normal, post‐irradiation and polycythemic mice using fluorescence‐activated cell sorting

Label-fracture: a method for high resolution labeling of cell surfaces.

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